Gravity sensing in plant and animal cells

Gravity determines shape of body tissue and affects the functions of life, both in plants and animals. The cellular response to gravity is an active process of mechanotransduction. Although plants and animals share some common mechanisms of gravity sensing in spite of their distant phylogenetic origin, each species has its own mechanism to sense and respond to gravity. In this review, we discuss current understanding regarding the mechanisms of cellular gravity sensing in plants and animals. Understanding gravisensing also contributes to life on Earth, e.g., understanding osteoporosis and muscle atrophy. Furthermore, in the current age of Mars exploration, understanding cellular responses to gravity will form the foundation of living in space

Enhancement of protein production via the strong DIT1 terminator and two RNA-binding proteins in Saccharomyces cerevisiae

Post-transcriptional upregulation is an effective way to increase the expression of transgenes and thus maximize the yields of target chemicals from metabolically engineered organisms. Refractory elements in the 3′ untranslated region (UTR) that increase mRNA half-life might be available. In Saccharomyces cerevisiae, several terminator regions have shown activity in increasing the production of proteins by upstream coding genes; among these terminators the DIT1 terminator has the highest activity. Here, we found in Saccharomyces cerevisiae that two resident trans-acting RNA-binding proteins (Nab6p and Pap1p) enhance the activity of the DIT1 terminator through the cis element GUUCG/U within the 3′-UTR. These two RNA-binding proteins could upregulate a battery of cell-wall–related genes. Mutagenesis of the DIT1 terminator improved its activity by a maximum of 500% of that of the standard PGK1 terminator. Further understanding and improvement of this system will facilitate inexpensive and stable production of complicated organism-derived drugs worldwide

Radiation Hybrid Maps of Medaka Chromosomes LG 12, 17, and 22

The Medaka is an excellent genetic system for studies of vertebrate development and disease and environmental and evolutionary biology studies. To facilitate the mapping of markers or the cloning of affected genes in Medaka mutants identified by forward-genetic screens, we have established a panel of whole-genome radiation hybrids (RHs) and RH maps for three Medaka chromosomes. RH mapping is useful, since markers to be mapped need not be polymorphic and one can establish the order of markers that are difficult to resolve by genetic mapping owing to low genetic recombination rates. RHs were generated by fusing the irradiated donor, OLF-136 Medaka cell line, with the host B78 mouse melanoma cells. Of 290 initial RH clones, we selected 93 on the basis of high retention of fragments of the Medaka genome to establish a panel that allows genotyping in the 96-well format. RH maps for linkage groups 12, 17, and 22 were generated using 159 markers. The average retention for the three chromosomes was 19% and the average break point frequency was ∼33 kb/cR. We estimate the potential resolution of the RH panel to be ∼186 kb, which is high enough for integrating RH data with bacterial artificial chromosome clones. Thus, this first RH panel will be a useful tool for mapping mutated genes in Medaka

Generation of medaka gene knockout models by target-selected mutagenesis

We have established a reverse genetics approach for the routine generation of medaka (Oryzias latipes) gene knockouts. A cryopreserved library of N-ethyl-N-nitrosourea (ENU) mutagenized fish was screened by high-throughput resequencing for induced point mutations. Nonsense and splice site mutations were retrieved for the Blm, Sirt1, Parkin and p53 genes and functional characterization of p53 mutants indicated a complete knockout of p53 function. The current cryopreserved resource is expected to contain knockouts for most medaka genes

WDR55 Is a Nucleolar Modulator of Ribosomal RNA Synthesis, Cell Cycle Progression, and Teleost Organ Development

The thymus is a vertebrate-specific organ where T lymphocytes are generated. Genetic programs that lead to thymus development are incompletely understood. We previously screened ethylnitrosourea-induced medaka mutants for recessive defects in thymus development. Here we report that one of those mutants is caused by a missense mutation in a gene encoding the previously uncharacterized protein WDR55 carrying the tryptophan-aspartate-repeat motif. We find that WDR55 is a novel nucleolar protein involved in the production of ribosomal RNA (rRNA). Defects in WDR55 cause aberrant accumulation of rRNA intermediates and cell cycle arrest. A mutation in WDR55 in zebrafish also leads to analogous defects in thymus development, whereas WDR55-null mice are lethal before implantation. These results indicate that WDR55 is a nuclear modulator of rRNA synthesis, cell cycle progression, and embryonic organogenesis including teleost thymus development

Microinjection of medaka embryos for use as a model genetic organism

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory

Essential techniques for introducing medaka to a zebrafish laboratory - Towards the combined use of medaka and zebrafish for further genetic dissection of the function of the vertebrate genome

The medaka, Oryzias latipes, a small egg-laying freshwater fish, is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens have been carried out. Despite a number of large-scale screens in zebrafish, a substantial number of mutants with new distinct phenotypes were identified in similar large-scale screens in the medaka. This observed difference in phenotype is due to the two species having a unique combination of genetic, biological and evolutional properties. The two genetic models share a whole-genome duplication event over that of tetrapods; however, each has independently specialized or lost the function of one of the two paralogues. The two fish species complement each other as genetic systems as straightforward comparison of phenotypes, ease of side-by-side analysis using the same techniques and simple and inexpensive husbandry of mutants make these small teleosts quite powerful in combination. Furthermore, both have draft genome sequences and bioinformatic tools available that facilitate further genetic dissection including whole-genome approaches. Together with the gene-driven approach to generate gene knockout mutants of the fish models, the two fish models complement the mouse in genetically dissecting vertebrate genome functions. The external embryogenesis and transparent embryos of the fish allow systematic isolation of embryonic lethal mutations, the most difficult targets in mammalian mutant screens. This chapter will describe how to work with both medaka and zebrafish almost as one species in a lab, focusing on medaka and highlighting the differences between the medaka and zebrafish systems. © 2011 Springer Science+Business Media, LLC